There are a number of albumin-free rFVIII formulations on the market using stabilization and formulation matrix excipients, such as, sodium chloride, glycine, mannitol, sucrose, trehalose, histidine, and calcium chloride. Calcium chloride is considered an essential component in the formulation because Factor VIII is a large hetero-dimer molecule where the heavy and light chains are held together by a metal ion bridge of divalent cations such as calcium and copper ions. All the current formulations contain calcium chloride, which has been assumed to be essential in order to maintain Factor VIII integrity and stability. The concentration of calcium chloride in the various formulations varies from 1.7 mM to 5 mM. It has been shown recently the calcium (Ca2+) is necessary for the generation of Factor VIII cofactor activity but that Ca2+ did not affect heavy chain-light chain binding affinity. It has been shown that Factor VIII contains two Ca2+ binding sites with different affinities and that the cofactor activity of Factor VIII can be reconstituted from heavy chain and light chain only when both chains are preactivated by Ca2+.